title: Circulating fragments of N-terminal pro-B-type natriuretic peptides in plasma of heart failure patients.
abstract: BACKGROUND
  The use of nonstandardized N-terminal pro-B-type natriuretic peptide (NT-proBNP) assays can contribute to the misdiagnosis of heart failure (HF). Moreover, there is yet to be established a common consensus regarding the circulating forms of NT-proBNP being used in current assays. We aimed to characterize and quantify the various forms of NT-proBNP in the circulation of HF patients.
  
  
  METHODS
  Plasma samples were collected from HF patients (n = 20) at rest and stored at -80 °C. NT-proBNP was enriched from HF patient plasma by use of immunoprecipitation followed by mass spectrometric analysis. Customized homogeneous sandwich AlphaLISA® immunoassays were developed and validated to quantify 6 fragments of NT-proBNP.
  
  
  RESULTS
  Mass spectrometry identified the presence of several N- and C-terminally processed forms of circulating NT-proBNP, with physiological proteolysis between Pro2-Leu3, Leu3-Gly4, Pro6-Gly7, and Pro75-Arg76. Consistent with this result, AlphaLISA immunoassays demonstrated that antibodies targeting the extreme N or C termini measured a low apparent concentration of circulating NT-proBNP. The apparent circulating NT-proBNP concentration was increased with antibodies targeting nonglycosylated and nonterminal epitopes (P < 0.05).
  
  
  CONCLUSIONS
  In plasma collected from HF patients, immunoreactive NT-proBNP was present as multiple N- and C-terminally truncated fragments of the full length NT-proBNP molecule. Immunodetection of NT-proBNP was significantly improved with the use of antibodies that did not target these terminal regions. These findings support the development of a next generation NT-proBNP assay targeting nonterminal epitopes as well as avoiding the central glycosylated region of this molecule.
authors.affiliation: The Australian Institute for Bioengineering and Nanotechnology.
authors.forename: Jared Yong Yang
authors.initials: JY
authors.lastname: Foo
authors.forename: Yunxia
authors.initials: Y
authors.lastname: Wan
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Schulz
authors.forename: Karam
authors.initials: K
authors.lastname: Kostner
authors.forename: John
authors.initials: J
authors.lastname: Atherton
authors.forename: Justin
authors.initials: J
authors.lastname: Cooper-White
authors.forename: Goce
authors.initials: G
authors.lastname: Dimeski
authors.forename: Chamindie
authors.initials: C
authors.lastname: Punyadeera
mesh_terms: D000368:Aged; D005260:Female; D006333:Heart Failure / Q000097:blood*; D006801:Humans; D007118:Immunoassay; D008297:Male; D020097:Natriuretic Peptide, Brain / Q000097:blood*; D010446:Peptide Fragments / Q000097:blood*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; C109794:pro-brain natriuretic peptide (1-76); D020097:Natriuretic Peptide, Brain